Immuno-modulatory composition

ABSTRACT

A composition for modulating the immune response in a mammal comprising a pharmaceutically acceptable carrier solution and a plurality of biodegradable nanoparticles, wherein the nanoparticles comprise a targeting moiety that is able to bind selectively to the surface of a T lymphocyte cell and/or of a vascular endothelial cell and wherein the nanoparticles further comprise leukaemia inhibitory factor (LIF). Nanoparticle-mediated targeted delivery of LIF can be used a means to guide tolerogenesis in a patient and has immediate clinical application for recipients of organ grafts and also for patients suffering from autoimmune disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/739,357 filed May 19, 2010, now U.S. Pat. No. 9,101,548, which is a371 of International Patent Application No. PCT/GB2008/003626 filed Oct.24, 2008, which claims priority to Great Britain Patent Application No.0721081.8 filed Oct. 26, 2007. These applications are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

The invention is in the field of compositions for modulating andcontrolling the immune response in an animal, such as a human. Alsoconcerned are methods for controlling immune response in an animal or intissues and cells derived from an animal and which are to be utilised incell and tissue transplantation.

BACKGROUND OF THE INVENTION

Immune-mediated diseases arise when errors occur within the immunesystem. Normally the immune response carries the potential to destroyforeign antigens whilst at the same time protecting against auto-immuneattack. This exquisite ability to discriminate between “self” and“non-self” is orchestrated by the T lymphocytes: these cells ensuredestructive aggressive activity only kills foreign targets whilst theimmune response to the host (“self”) is actively protective andtolerant.

Dominant antigen-specific tolerant T lymphocytes include T regulatorycells (T_(reg)), that perform an important role in moderating the immuneresponse in an organism so as to maintain self-tolerance. Cellsconforming to the T_(reg) phenotype are a specialized group of Tlymphocytes that express cell surface markers including CD4, CD25,CTLA-4, and GITR. T_(reg) cells express the transcription factor Foxp3.Foxp3 functions as a transcriptional repressor required for lymphocytedevelopment down the T_(reg) lineage and loss of Foxp3 functiontypically results in early death in humans due to the over-whelmingauto-immune disease known as IPEX (immunodysregulationpolyendocrinopathy enteropathy X-linked syndrome). T_(reg) cells arepredominantly derived from the thymus, where they develop under arigorous process of selection to ensure their self-tolerant reactivityprior to release into wider circulation. Circulating T_(reg) cellscontribute to down-regulation of the body's aggressive immune responsesagainst foreign pathogens. T_(reg) cells also dominate and suppress anyaggressive immune responses that may otherwise lead to attack of thebody's own tissues (i.e. self tolerance).

Disease can ensue when self-tolerant T_(reg) cells become weakened,thereby allowing aggressive cells to break through. In some individualscrippling auto-immune diseases will develop. Such inappropriate immuneactivity is thought to underly a wide number of diseases that lackcurative therapy including rheumatoid arthritis, type I diabetesmellitus, systemic lupus erythematosis (SLE), psoriasis, and Crohn'sdisease to name a few.

Immune tolerance is also of crucial importance in the field of organtransplantation. There is a critical shortage of donor organs and it isof primary concern that appropriate tissue typing is conducted on thedonor tissue and the recipient prior to transplantation. In spite of anapparently good match between a recipient host and a donor, therecipient's immune system will naturally recognize the allograftedtissue as ‘foreign’ and if left uncontrolled will reject the transplant.Consequently, donor organ recipients must remain on immunosuppressivetherapy for the remainder of their lives following transplant. Eventhen, recipients are subject to progressive chronic rejection within theblood vessels of the transplanted organ due to an insidious process thatis not controlled by current immunosuppressive drugs: such chronicvascular rejection eventually blocks the blood flow within thetransplanted organ causing organ failure. Without a new organtransplant—unlikely given the shortage of organs—or dialysis in the caseof kidney graft recipients, the patient will die.

In 2004 around 15,000 kidney transplant procedures were carried out inthe US alone, with a one year graft survival rate of around 90% (fromOPTN/SRTR Data as of May 1, 2006). Nevertheless, at the filing date ofthis application over 70,000 people were on the waiting list for akidney transplant in the US, clearly demonstrating that demand outstripssupply many times over. For other organs the graft survival rate falls,for example the one year survival rate for liver transplants was around83%, and the demand also is greater than the available supply of donormaterial. Apparently high graft survival rates often belie the fact thattransplantation is often only available for individuals with a highlyfavorable clinical case. Those patients who are unfortunately regardedas being poorer candidates for transplant will rarely ever becomerecipients of much needed donor tissue.

There is a need to improve control of the immune response to promotehost tolerance to allografted tissue such that host acceptance oftransplanted material is increased. In addition, modulation of theimmune response in the recipient so as to promote recognition of theforeign transplanted tissue as ‘self’ may also serve to reduce thedependency on lifelong immunosuppressive therapy. Further, improvedimmunomodulatory treatments may also effect the ability to match donortissue with recipients in need of a transplant, by expanding theparameters for tissue type matching—i.e. by expanding the toleranceparameters for mismatch between the tissue type of the host and that ofthe donor. This is of primary importance for patients with rare tissuetypes, such as those from particular ethnic minorities.

The present invention seeks to overcome or at least reduce the problemsthat exist by providing compositions and methods for targeted modulationof the immune response and promoting proliferation and activity ofT_(reg) cells that will suppress the anti-donor response and/or thechronic vascular rejection process. Not only does the targeted mode ofdelivery bring immune-modulators to the site where they are needed, forexample within the organ transplant, but also it reduces the overallexposure of the patient to bio-active components that may carry toxicside-effects at irrelevant sites.

SUMMARY OF THE INVENTION

The invention provides directed therapy specifically to the site ofimmune activity that is associated with the diseased state. Theinvention also exploits a novel molecular approach to reduce aggressiveimmune activity by harnessing the body's own protective mechanism. Theclinical benefit is considerable both for patients with autoimmunedisease and for organ graft recipients.

A first aspect of the invention provides a composition for modulatingthe immune response in a mammal comprising:

-   -   a) a pharmaceutically acceptable carrier solution; and    -   b) a plurality of biodegradable nanoparticles, wherein the        nanoparticles comprise a targeting moiety that is able to bind        selectively to the surface of a T lymphocyte cell and/or of a        vascular endothelial cell and wherein the nanoparticles further        comprise leukaemia inhibitory factor (LIF).

A second aspect of the invention provides a method of inducing a Tlymphocyte cell to adopt a T_(reg) phenotype comprising exposing the Tlymphocyte to a plurality of biodegradable nanoparticles, wherein thenanoparticles comprise a targeting moiety that is able to bindselectively to the surface of the T lymphocyte cell and/or to a vascularendothelial cell and wherein the nanoparticles further compriseleukaemia inhibitory factor (LIF).

A third aspect of the invention provides a biodegradable nanoparticlecomprising a biodegradable carrier material that encapsulates atherapeutic compound, a therapeutic compound, and a surface locatedtargeting moiety, characterised in that the therapeutic compound is LIFand the surface located targeting moiety is an antibody, or an antigenbinding fragment of an antibody, that specifically binds to an antigenpresent on the cell surface of a T lymphocyte and/or of a vascularendothelial cell.

A fourth aspect of the invention provides use of a preparationcomprising a plurality of biodegradable nanoparticles, wherein thenanoparticles comprise a targeting moiety that is able to bindselectively to the surface of a T lymphocyte cell and/or of a vascularendothelial cell, and wherein the nanoparticles further compriseleukaemia inhibitory factor (LIF), in the manufacture of a compositionfor moderating immune response in a patient. Other aspects of theinvention provide uses for the treatment of autoimmune disease and graftversus host disease.

A further aspect of the invention provides a method of promoting animmune tolerance response in a patient comprising administering to thepatient a therapeutically effective amount of a composition comprising aplurality of biodegradable nanoparticles, wherein the nanoparticlescomprise a targeting moiety that is able to bind selectively to thesurface of a T lymphocyte cell and/or of a vascular endothelial cell,and wherein the nanoparticles further comprise leukaemia inhibitoryfactor (LIF).

A further aspect of the invention provides a method of storing mammaliantissue destined for allografting into a recipient comprising perfusingthe tissue with a solution that comprises a plurality of biodegradablenanoparticles, wherein the nanoparticles comprise a targeting moietythat is able to bind selectively to the surface of a T lymphocyte celland/or of a vascular endothelial cell, and wherein the nanoparticlesfurther comprise leukaemia inhibitory factor (LIF).

These and other aspects and embodiments of the invention will becomeapparent from the detailed description of preferred embodiments of thepresent invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagrammatic cross sectional view of an exemplary Tlymphocyte or vascular endothelial cell targeted nanoparticle of theinvention, wherein the nanoparticle in the construction shown in FIG. 1is a LIF-loaded, avidin-coated nanoparticle precoated with biotinylatedantibody.

FIG. 2 (a) shows the results of an experiment in histogram format inwhich T lymphocytes from axotrophin null mutant mice are treated withLIF and then assayed for release of the pro-inflammatory cytokineinterferon gamma (LIF is shown in black the control is shown inhatching).

FIG. 2 (b) shows interferon gamma release is very high in rejection andis partially inhibited by LIF. Release of interferon gamma into thesupernatant of primed rejected cultures increased with time, reaching 16ng/ml at 5 days after reboost with donor antigen; the presence ofrecombinant LIF suppressed this interferon gamma release by around 50%.In tolerance, interferon gamma never exceeded 200 pmol, with or withoutLIF.

FIG. 3 shows the results of an experiment in which genetically normalmouse spleen cells were activated by CD3/CD28 ligation in the presenceor absence of exogenous LIF.

FIG. 4 shows the results of an experiment in histogram format in whichspleen cells from in vivo primed allo-tolerant mice stimulated withdonor antigen (black bars) display increased levels of LIF protein anddecreased levels of IL6 protein when compared to spleen cells from invivo primed allo-rejected mice stimulated with donor antigen (whitebars), indicating that LIF protein is linked to allo-tolerance and IL6protein is linked to allo-rejection.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides nanoparticle-mediated targeted delivery of LIF(leukaemia inhibitory factor) as a novel means to guide tolerogenesis ina patient. The inventors have previously shown that LIF is a keyregulator of the immune response in which T_(reg) lymphocytescollectively ensure protective tolerance towards self-tissues whilstsimultaneously enabling aggressive attack towards foreign pathogens.Without being bound by theory, the role of LIF in this critical balanceis believed to be linked to T_(reg) cells and acts in concert withFoxp3. Sustained LIF activity is an intrinsic feature of T_(reg) cells,supporting the notion that LIF provides a cue for T_(reg) developmentand T_(reg) maintenance. The present invention utilises LIF-loadednanoparticles that are specifically targeted to T lymphocytes so as toguide naïve T cells into becoming antigen-specific T_(reg) cells viaexposure to high local concentrations of LIF activity. An advantage ofthis approach is that it has immediate clinical application forrecipients of organ grafts and also for patients suffering fromautoimmune disease.

According to the present invention modulation of immune tolerance isconsidered to encompass a suppression of an organism's inherent basallevel of immune function. Typically, the organism is an animal, moretypically a mammal or a human. The basal level is taken as the level ofimmune reactivity in the organism prior to treatment with a compositionof the invention. In a normal healthy individual, such as a humanpatient, the immune level would be equivalent to that of an unchallengedsteady-state level. Whereas in a patient suffering from an autoimmunedisorder the basal immune response would be elevated in comparison witha normal healthy patient. Likewise, in a patient who has recentlyreceived allografted tissue, one would expect to see an elevated basallevel of immune activity in the absence of immunosuppressive therapysuch as cyclosporine. Elevated immune activity can be identified by arelative increase in aggressive killer white blood cells such asneutrophils, macrophages, and cytotoxic T cells, as well as elevatedlevels of cytokines such as IL-2, IL-6, IL-13, IL-17, IL-23, andinflammatory mediators including interferon-gamma, TNFα and IL1β.

LIF is a member of the IL-6 family of cytokines. It is a secretedsignalling factor that binds to and signals via a LIF-specific cellsurface receptor gp190 that interacts with the gp130 signal transducingreceptor. Downstream intracellular signal propagation occurs via theJAK/STAT pathway, especially via the transcription factor STAT-3. LIFsignalling activity is terminated by SOCS3, SOCS3 being induced in afeedback loop mechanism. The present inventors have previouslyidentified that ex vivo treatment of murine spleen cells with LIFincreases Foxp3 transcription. Further, in contrast to a mutant FOXP3that is unable to bind DNA and function as a transcriptionalrepressor—wild-type FOXP3 strongly inhibits SOCS-3 (suppressor ofcytokine signalling-3) (Muthukumarana et al 2007). Since SOCS-3 is afeedback inhibitor of LIF signalling, its repression by wild-type FOXP3in T_(reg) cells will have a positive effect on LIF activity. In T_(reg)cells LIF signalling activity is also known to be linked to activity ofthe E3 ligase MARCH VII (axotrophin) which in-turn regulates activity ofFoxp3. A hierarchical regulatory cassette between LIF, Foxp3, andaxotrophin is envisaged. Given that Foxp3 is considered to be one of thearchetypal markers of the T_(reg) phenotype, regulation of LIF activityin the T_(reg) cells and in T_(reg) precursor cells can have a profoundeffect upon the tolerogenic immune response in an animal.

The pleiotrophic properties of LIF are wide ranging and include thecardiovascular system. Targeted over-expression of LIF in infarctedmyocardium prevents myocardial loss and improves post-infarct cardiacfunction in rats (Berry M F et al. J Thorac Cardiovasc Surg. 2004December; 128(6):866-75). LIF also displays a multifaceted capacity fortreating occlusive vascular disease, particularly at the early stages ofatherosclerotic plaque formation (Moran C S et al. Arterioscler Thromb.1994 August; 14(8):1356-63; World C J et al. Ann N Y Acad Sci. 2001December; 947:323-8) and LIF gene therapy has been shown to bebeneficial for skin allograft survival in mice (Akita S. et al.Transplantation. 2000 October 15; 70(7):1026-31).

In an embodiment, the present invention provides compositions comprisingLIF that can be targeted to a location on a cell, within a tissue orwithin the body of a patient or animal. For example, compositions of theinvention can comprise bioavailable LIF that is directed to cells thatexpress the cell surface receptor CD4 (CD4+ cells) and thereby increaselocalised exposure to LIF for these cells. In another embodiment of theinvention LIF can be targeted to cells that express a cell surfacebiomarker that is characteristic of or associated with pluripotency,such as stage specific embryonic antigen 1 (SSEA-1). Other cell surfaceexpressed molecules that could be targeted by the compositions of theinvention include VEGF Receptor, EPCR, CD34 and/or CD31 expressed onvascular endothelial cells to generate a tolerogenic micro-environmentwithin an organ or tissue.

In accordance with the invention, T cells that are uncommitted can bediverted down the path to becoming T_(reg) (self-tolerant) cells byexposure to LIF signalling. LIF cannot be readily administeredsystemically due to its cytotoxicity and short half-life in vivo. Thepresent invention provides a targeted approach to LIF delivery, whichprovides the dual advantages of increasing the local concentration ofLIF at the point of need (i.e. at the cell surface of theantigen-responsive T cell) and reducing the overall concentration of LIFthat is required to obtain the desired therapeutic effect. In a specificembodiment of the invention, LIF containing nanoparticles are targetedat T lymphocytes, in particular T lymphocyte specific markers located onthe cell surface. Alternatively, LIF containing nanoparticles aretargeted to vascular endothelial cells. Targeting to the specified cellsurface marker is typically achieved by locating a targeting moiety,such as antibodies, on the surface of the nanoparticle. Polymer basednanoparticles that comprise antibody targeting moieties are described inWO-A-2006/080951, liposomal nanoparticles that comprise antibodytargeting moieties are described in WO-A-2005/051305.

As used herein, the term ‘antibody’ denotes a protein that is producedin response to an antigen that is able to combine with and bind to theantigen, preferably at a specific site on the antigen, known as anepitope. The term as used herein includes antibodies of polyclonal andmonoclonal origin, unless stated otherwise. Polyclonal antibodies are agroup of antibodies produced by different B lymphocytes in response tothe same antigen; different antibodies in the group typically recognizedifferent parts (epitopes) on the antigen. A monoclonal antibodyrecognizes only one type of antigen and is produced by the daughtercells of a single antibody-producing lymphocyte, typically a hybridoma.Also included within the term ‘antibody’ are antigen binding fragmentsof naturally or non-naturally occurring antibodies, for example, the“Fab fragment”, “Fab′ fragment” (a Fab with a heavy chain hinge region)and “F(ab′)2 fragment” (a dimer of Fab′ fragments joined by a heavychain hinge region). Recombinant methods have been used to generatesmall antigen-binding fragments, such as “single chain Fv” (variablefragment) or “scFv,” consisting of a variable region light chain andvariable region heavy chain joined by a synthetic peptide linker. Unlikeantibodies derived from other mammals, camelid species express fullyfunctional, highly specific antibodies that are devoid of light chainsequences. Camelid heavy chain antibodies are of particular use, as theyare found as homodimers of a single heavy chain, dimerized via theirconstant regions. The variable domains of camelid heavy chain antibodiesare referred to as VHH domains and retain the ability, when isolated assmall fragments of the VH chain, to bind antigen with high specificity(Hamers-Casterman et al., 1993, Nature 363: 446-448; Gahroudi et al.,1997, FEBS Lett. 414: 521-526). Further included within the term‘antibody’ are so called camelized mutants of human VH domains thatretain antigen binding activity but exhibit some of the advantages ofcamelid VHH domains (Riechmann, 1994, FEBS Lett. 339: 285-290). Inaddition to antigen binding fragments, antibodies of the presentinvention can include derivatives of antibodies, such as chimericfusions with labelling moieties including green fluorescent protein(GFP).

Alternative non-antibody targeting moieties can be utilised to targetthe nanoparticles of the invention to the cells of choice. For example,affinity labels such as streptavidin can be used to target abiotinylated target. Other suitable affinity labels will be known in theart and can include tethered ligands for a cell surface receptor knownto be specifically expressed on the target cell.

In an embodiment of the invention the composition comprises abiodegradable polymer based nanoparticle comprising encapsulated LIFpolypeptide, which nanoparticle further comprises surface exposedantibody that specifically binds for example to the CD4 receptor.Suitably, the polymer is a polylactide-co-glycolide polymer (PLG). Thenanoparticles of the invention are resuspended in a biocompatiblesolution, such as phosphate buffered saline, and can be used in vivo(e.g. via parenteral administration), in vitro or ex vivo in order tomodulate the immune response. Ex vivo uses can include pre-treatment ofdonor tissue intended for allografting with the compositions of theinvention shortly before implant, thereby improving the chances ofacceptance of the allografted tissue by the recipient. In this latterembodiment of the invention, the nanoparticles can comprise analternative targeting moiety in addition to or in place of theCD4-binding antibody, for example an antibody that targets theendothelial cells present within the allograft tissue. In such a way,LIF can be controllably released from within the allografted tissue inthe period immediately after transplant, thereby facilitating the immunetolerance reaction in the recipient.

The compositions of the invention are suitable for the treatment ofautoimmune disorders. Local parenteral or subcutaneous delivery of thenanoparticle-comprising compositions of the invention into or around aninflamed joint can assist in amelioration of the symptoms of rheumatoidarthritis. Also, topical administration of an appropriate nanoparticlecomposition (i.e. as a lotion or skin cream) can be effective intreatment psoriasis. Finally, the nanoparticles can be incorporated intoa time release depot formulation for longer term use, suitable fortreatment of auto-immune encephalopathies and multiple sclerosis.

The compositions of the invention are useful for the control of immuneresponse in vivo, ex vivo or in vitro. In a specific embodiment of theinvention tissues and organs intended for transplantation (e.g. heart,kidney, bone and blood vessel grafts) can be pre-treated with thecompositions of the invention prior to implantation into the recipient.Cellular allograft tissue, such as bone marrow or stem cells can also betreated in vitro or ex vivo, prior to introduction into the recipient.As such, the compositions of the invention provide a facilitatingtechnology for the expansion of a variety of regenerative medicinetherapies.

In a particular embodiment, nanoparticles of the invention are comprisedwithin the perfusion solution that is used for organ and tissuepreservation during transplant of donor organs. In this way, the LIFcomprised within the nanoparticles serves a dual purpose of maintainingproliferation of endogenous stem cells within the transplant tissue aswell as favouring immune tolerance in the recipient after the transplantprocedure has been completed.

Example 1

Experimental models in mice have identified a critical regulatory systemin T lymphocytes wherein Foxp3—the master gene for regulatorytolerance—is itself regulated by axotrophin/MARCH-7 (an E3-ligase) andLIF. Not only does axotrophin/MARCH-7 directly regulate LIF release by Tlymphocytes, but also both axotrophin/MARCH-7 and LIF are required fornormal Foxp3 gene activity.

In humans, Foxp3 and axotrophin/MARCH-7 are co-expressed in peripheralblood cells. In patients who have received a bone marrow transplant, andalso in patients who have received a kidney transplant, expression ofFoxp3 and axotrophin/MARCH-7 positively correlate with good graftfunction (Muthukumarana et al 2007). These findings infer that therelationship between immune tolerance, axotrophin/MARCH-7 and—byextrapolation—LIF is valid in clinical patients and supports the presentinvention which adopts a novel tolerogenic therapeutic approach oftargeted delivery of LIF to the site of immune activity.

Reciprocity Between Foxp3 and LIF

TABLE 1 SOCS-3: fold change from 0 h to 24 h after induction of Foxp3 inhuman T cells transcript Wild-type FOXP3 DE251 FOXP3 SOCS-3 12.00 345.00LIF 64.00 0.02 FOXP3* 9.00 10.00 *both transcript and protein equivalent

Targeted Drug Delivery Using Nanoparticles

Polymeric biodegradable nanoparticles that combine high drug loadingwith targeting to specific cell types have been developed by YaleUniversity (see WO-A-2006/080951; and Fahmy et al 2005). LIF-loadednanoparticles were prepared according to this protocol: these werecoated with avidin to permit addition of biotinylated antibody. Thepresent inventors have extended the use of this therapeutic resource tocontrol the immune response for transplant recipients and for patientswith auto-immune disease.

In accordance with the present invention, it is considered that LIF is apreviously unrecognised regulator of immune tolerance and functions inconcert with Foxp3. Accordingly it is proposed that LIF therapy targetedto CD4+ lymphocytes will guide naïve T cells towards the tolerantphenotype (T_(reg)).

Experimental Design:

Based on clinical, experimental, and molecular data this Example tests anovel therapeutic approach that combines two strategies, namely (i)targeted delivery and (ii) harnessing of natural regulatory pathways.

Interferon Gamma Release is Reduced in Immune Cells Exposed to LIFTreatment

Interferon gamma release is strongly correlated with rejection ex vivo,whilst self-self controls reveal low background levels of interferongamma. When stimulated by a third party antigen (C57B16) tolerant andrejected cultures of CBA spleen cells from a BALB/c tolerant orrejection mouse model each release high levels of interferon gamma butwith slower release kinetics when compared to primed cultures,demonstrating the specificity of the allo-tolerant ex vivo state whereinterferon gamma is low. When serum-free cell cultures of these cellsare treated with LIF the release of interferon gamma in primed rejectionis markedly reduced, this being halved from control values of around 9ng/ml to around 4 ng/ml (FIG. 2 (b)). In primed tolerant cellsinterferon gamma levels are very low, and largely unresponsive toaddition of exogenous LIF. The concentration of released interferongamma is determined according to ELISA and Western blotting.

The suppressive effect of LIF signalling on interferon gamma release wasincreased to 90% suppression when naïve spleen cells taken fromaxotrophin null mutant mice were activated in vitro by CD3/CD28cross-linking (FIG. 2( a)). This indicates that LIF treatment can leadsto suppression of release of the inflammatory cytokine interferon gammafrom immune cells in vitro under certain cellular conditions.

LIF Therapy Using Nanoparticles

Since T_(reg) cells arise from CD4+ T cells the LIF-loaded nanoparticlesare precoated with biotinylated-anti-CD4 antibody (as shown in FIG. 1)the aim being to target LIF to the sites of antigen engagement for CD4+lymphocyte activation. Unloaded nanoparticles act as controls whilstdelivered LIF dose is determined by comparison between treated andcontrol supernatants from the in vitro experiments.

The strategy has the additional benefit that nanoparticles may also bephagocytosed by antigen presenting cells, further increasing thedelivery of LIF to the micro-environment of the responding lymphocyte.In vivo, tissue uptake of the nanoparticles from blood is measured inall the major organs including lung, liver, kidney, spleen, lymph node,thymus, heart, and brain. Nanoparticle size of <500 nm can reducesequestration to the liver: should this remain a problem “stealth”nanoparticles can be prepared by including biotinylated poly-ethyleneglycol (PEG) in the surface coat.

Foxp3 Measurement by Flow Cytometry

A critical readout for the experiments is expression of Foxp3 protein inCD4+ lymphocytes (see FIG. 2). Standardised flow cytometry protocolemploys six colour analyses of single cells in complex populations:accordingly each cell is characterised against 36 potential variables.Surface staining includes for CD4, CD3, CD25, CD8, CTLA-4, and gp190(the LIF-specific receptor subunit) whilst intracellular stainingmeasures Foxp3 and cytoplasmic CTLA-4. Cell populations are phenotypedat 0 h and after culture plus stimulating allo-antigen in the presenceor absence of LIF nanoparticles. In accordance with the methods of thepresent invention, LIF nanoparticle therapy leads to a significantincrease in the numbers of Foxp3+ cells.

Three experimental approaches are taken to validate the results further.

(i) Mixed Lymphocyte Response:

An in vitro model of allo-reactivity is the one way mixed lymphocyteresponse (MLR) and human peripheral blood lymphocytes are used inestablished MLR protocols to measure both DNA synthesis by tritiatedthymidine uptake and expression of Foxp3 by single cell flow cytometricanalysis following activation in the presence or absence of LIF therapy.These experiments can identify the effect of LIF during priming toallo-antigen over a 7 day period.

(ii) Primed MLR Response:

Use of allo-reactive primed lymphocytes asks to what extent can primeddonor-specific immune reactivity be guided towards the tolerant state byLIF therapy. Here both human and murine models are used. Humanexperiments look at peripheral blood lymphocytes following stimulationwith commercially available “donor-specific” antigen coated beadsfollowed by re-boosting with beads used in the priming step, or withthird-party beads. Parallel ex vivo studies utilise CBA mice groupedinto “allo-tolerant” versus “allo-rejected” recipients of a fullymismatched BALB/c heart allograft: here donor-antigen stimulation is viairradiated BALB/c spleen cells using standard protocols Again it isdetermined whether LIF-loaded nanoparticles bias the allo-rejectedanti-donor response towards tolerance, by looking for increases in Foxp3positive cell numbers over a 7 day period. Full phenotype profilingoccurs at time zero, 3, 5, and 7 days to look for changes asallo-activation progresses.

(iii) In Vivo Tolerogenesis by LIF Therapy:

To be able to influence T_(reg) development in vivo, rather than inculture, represents a significantly greater challenge since it is knownthat recombinant LIF delivered intravenously or into the peritoneum isvery rapidly degraded [Hilton et al 1991]. The present inventors havedeveloped a novel approach using targeted nanoparticles that representsa powerful means to achieve LIF delivery direct to the micro-environmentof naïve T cells, where slow release will optionally be continuous overseveral weeks, and the murine heart allograft model is suitable fortesting the efficacy of such LIF therapy in vivo, typically as apreliminary to pre-clinical studies.

To test the effect of nanoparticle-encapsulated LIF on heart allograftsurvival in BALB/c mice receiving a vascularised CBA heart graft thefollowing trial has been devised. Recipient mice receive CD4 targetednanoparticle-encapsulated LIF (supplied by Tarek Fahmy and Jason Park,Yale University). Historical controls received no therapy and rejectedtheir graft around day 7 post-grafting. The importance of thisexperiment is the understanding of the regulatory mechanisms thatcontrol T cells in vivo.

Materials and Methods:

CBA to BALB/c heart grafts day 0: N=2

Micro-Encapsulated LIF Preparation:

Pre-zap all inert surfaces to eliminate static charge

6 mg nanoparticles in Eppendorf tube [˜6 micrograms LIF]

resuspend in 500 microlitres sterile PBS

Add 100 microlitres sterile biotinylated anti-CD4 stock (eBioscience CatN 36-0041 (13-0041) 0.5 mg in PBS no NHN₃:rat IgG2b, k) @ 1 mg/ml (0.1mg total)

-   -   Incubate at room temperature for 30 minutes    -   Spin @ 10,000 g for 5 minutes    -   Keep supernatant

Resuspend nanoparticles vigorously by sonication

-   -   WASH: Add slowly 1 ml sterile PBS    -   Spin @ 10,000 g for 5 minutes    -   Keep supernatant [˜3 micrograms LIF (assuming 50% loss)]

Treatment of Allograft Recipients:

Resuspend the nanoparticle pellet vigorously by sonication

-   -   Add slowly—with sonication—400 microlitres sterile PBS    -   Resuspend vigorously by sonication for intravenous delivery PER        MOUSE of 200 microlitres

Treatment is carried out on Day 0; Day 2; Day 4 by introducing 200microlitres of resuspended LIF nanoparticles into the tail vein using a27½ gauge needle.

At time of rejection (cessation of transplanted heart beat) the animalis culled and the donated heart is removed for analysis. The nativeheart is also preserved for analysis. Samples are also taken of blood,lung, liver, kidney, skin, thymus, lymph node, brain, large and smallintestine to determine nanoparticle distribution and uptake.

Mice

Gene trap insertion was used to generate axotrophin null BALB/c mice andlittermates from heterozygous parents were genotyped by PCR analysis ofgenomic DNA to identify axot+/+, axot+/−, and axot−/− pups as detailedpreviously. Spleen, thymus and lymph node were obtained from 5 m oldlittermates and kept on ice prior to cell preparation for the analysesdescribed below. The lymph node tissue yielded very few cells and wasdiscarded

Cell Preparations

Splenocytes and thymocytes were teased out from each organ and collectedin sterile growth medium [RPMI-1640 (Gibco™ Invitrogen Co.) supplementedwith 10% FCS (Gibco™ Invitrogen Co.), 200 mM L-Glutamine, 100 U/mLPenicillin and 100 μg/mL Streptomycin (Sigma Chemical Co.)]. The cellsuspensions were washed, resuspended in growth medium and counted usinga haemocytometer.

ELISA

ELISA's were performed on the 48 h culture supernatants, in 96-wellFalcon® plates using the DuoSet® ELISAS for Interferon gamma (DY485),IL2 (DY402), IL4 (DY404), IL10 (DY417) and Quantikine®M Immunoassay forLIF (MLF00), from R&D Systems. The standard curves were established byprocessing the optical density data using Microsoft Excel software andcytokine concentrations were determined using the standard curves.

Flow Cytometry

The splenic and thymic cell suspensions were RBC depleted and washed inFACS staining solution (0.2% BSA and 0.1% sodium azide in 1×PBS) priorto being mixed with the various monoclonal antibodies detailed below,these being either directly or indirectly conjugated with Phycoerythrin(PE) or Fluorescein isothiocyanate (FITC). PE-rat anti-mouse CD19(557399), PE-hamster anti-mouse TCRα chain (553172) and rat anti-mousedendritic cell clone 33D1 (551776) were from Pharmingen. Rat anti-mouseCD205-FITC (MCA949F), mouse anti-rat IgG2a heavy chain-FITC (MCA278F)and mouse anti-rat IgG2b chain-FITC were from Serotec Ltd. while rabbitanti-mouse CD25 (IL2Rα) and goat anti-rabbit IgG (H&L)-PE (4050-89) werefrom Santa Cruz Biotechnology and Southern Biotechnology Associatesrespectively. Anti CD4 (YTS177.9.6) and anti CD8 (YTS 105.18.10) were agift from Professor Stephen Cobbold, University of Oxford. Analyses wereperformed on a Becton Dickinson FACSCalibur instrument equipped withCellQuest software.

Example 2

This Example shows the effect of exogenous LIF on T cells activated byCD3/CD8 ligation.

Genetically normal mouse spleen cells cultured in serum-free growthmedium were activated by anti-CD3 and anti-CD28 in the presence orabsence of 10 ng/ml LIF. As shown in FIG. 3, at 48 h exogenous LIF wasfound to have increased expression of Nanog, p53, and LIF genes, anddecreased SOCS-3 gene expression.

It is therefore predicted that in vivo therapy using targeted deliveryof LIF will similarly increase the expression of Nanog, p53, and LIFgenes in a target cell, and depress SOCS-3 expression.

Example 3

This Example shows that cell-derived LIF protein is linked toallo-tolerance, whereas release of IL6 protein is linked toallo-rejection.

Spleen cells from in vivo primed allo-tolerant, or allo-rejected, micestimulated by donor antigen (i.e. irradiated donor-type spleen cells) inRPMI growth medium containing 10% FCS revealed that release ofcell-derived LIF protein is linked to allo-tolerance, whereas release ofIL6 protein is linked to allo-rejection (see FIG. 4).

It is therefore predicted that targeted delivery of LIF to CD4+ T cellsin vivo will result in increases in the regulatory tolerant (Treg)lineage, whereas targeted delivery of IL6 will oppose induction oftolerance.

CONCLUSION

The invention represents a highly innovative approach to treatment ofimmune-mediated disease using targeted LIF therapy. The experimentsincorporate two major advances of central relevance to treatment ofimmune-mediated indications, i.e. targeting plus harnessing of a naturaltolerogenic pathway, and thereby underpin development of new andsuccessful clinical therapies.

1. A composition for promoting an immune tolerance response in a mammalcomprising: a) a pharmaceutically acceptable carrier solution; and b) aplurality of biodegradable polylactidecoglycolide (PLG) polymernanoparticles, and wherein the PLG nanoparticles further comprise: (i)leukaemia inhibitory factor (LIF), wherein the LIF is encapsulated bythe PLG nanaoparticle; and (ii) the PLG nanoparticles comprise an outersurface, wherein a targeting moiety that is able to bind selectively toan antigen present on the surface of a T lymphocyte cell is linked tothe outer surface of the PLG nanoparticles.
 2. The composition of claim1, wherein the targeting moiety comprises a monoclonal antibody.
 3. Thecomposition of claim 1, wherein the targeting moiety comprises apolyclonal antibody.
 4. The composition of claim 1, wherein thetargeting moiety comprises an antigen-binding antibody fragment.
 5. Thecomposition of claim 4, wherein the antigen-binding antibody fragment isselected from the group consisting of: an Fab fragment; an Fab′fragment; an F(ab′)2 fragment; an scFv fragment.
 6. The composition ofclaim 1, wherein the targeting moiety binds specifically to an antigenpresent on the surface of the T lymphocyte cell, selected from the groupconsisting of: a CD4 receptor; a CD25 receptor; a gp190 receptor; a LIFreceptor.
 7. The composition of claim 1, wherein the targeting moiety isan antibody that specifically binds to a CD4 receptor present on thecell surface of the T lymphocyte cell.
 8. A composition for promoting animmune tolerance response in a mammal comprising: a) a pharmaceuticallyacceptable carrier solution; and b) a plurality of biodegradablepolylactidecoglycolide (PLG) polymer nanoparticles, and wherein the PLGnanoparticles further comprise: (i) leukaemia inhibitory factor (LIF),wherein the LIF is encapsulated by the PLG nanaoparticle; and (ii) thePLG nanoparticles comprise an outer surface, wherein a targeting moietythat is able to bind selectively to an antigen present on the surface ofa vascular endothelial cell is linked to the outer surface of the PLGnanoparticles.
 9. The composition of claim 8, wherein the targetingmoiety comprises a monoclonal antibody.
 10. The composition of claim 8,wherein the targeting moiety comprises a polyclonal antibody.
 11. Thecomposition of claim 8, wherein the targeting moiety comprises anantigen-binding antibody fragment.
 12. The composition of claim 11,wherein the antigen-binding antibody fragment is selected from the groupconsisting of: an Fab fragment; an Fab′ fragment; an F(ab′)2 fragment;an scFv fragment
 13. The composition of claim 8, wherein the targetingmoiety binds specifically to an antigen present on the surface of thevascular endothelial cell, selected from the group consisting of: a CD31receptor; a CD34 receptor; a VEGF receptor and a EPC receptor.
 14. Thecomposition of claim 1, wherein the biodegradable polylactidecoglycolidedegrades at a rate that allows for controlled release of theencapsulated LIF.
 15. The composition of claim 8, wherein thebiodegradable polylactidecoglycolide degrades at a rate that allows forcontrolled release of the encapsulated LIF.
 16. A composition forpromoting an immune tolerance response in a mammal comprising: a) apharmaceutically acceptable carrier solution; and b) a plurality ofbiodegradable polylactidecoglycolide (PLG) polymer nanoparticles, andwherein the PLG nanoparticles further comprise: (i) leukaemia inhibitoryfactor (LIF), wherein the LIF is encapsulated by the PLG nanaoparticles;and (ii) the PLG nanoparticles comprise an outer surface, and wherein abinding moiety is located on the outer surface.
 17. The composition ofclaim 16, wherein the binding moiety comprises avidin.
 18. Thecomposition of claim 16, wherein the binding moiety comprisesstreptavidin.